JP2023525898A - Use of Chelating Agents to Prevent Formation of Visible Particles in Parenteral Protein Solutions - Google Patents

Abstract

The present invention provides methods for preventing the formation of visible particles in aqueous protein formulations, in particular the use of certain chelating agents, and compositions and pharmaceutical products obtained by said methods. TIFF2023525898000021.tif96161

Description

The present invention relates to the field of aqueous protein compositions stabilized against the formation of visible particles, especially pharmaceutical antibody formulations for parenteral application.

BACKGROUND OF THE INVENTION Formation of visible particles during shelf life is one of the major concerns associated with biopharmaceuticals for parenteral use. Although the extent of clinical consequences is still not fully known, the presence of particles is generally considered a potential safety risk to patients and is therefore one of the most common reasons for parenteral product recall events. (Doessegger et al., 2012).

One of the most common underlying causes of particle formation in biopharmaceutical formulations is the degradation of polysorbates (PS), such as PS20, which are commonly added to formulations to protect proteins against interfacial stress. It is Polysorbates can be described as heterogeneous mixtures of partial esters of fatty acids with ethoxylated sorbitol or isosorbitol (Hewitt et al., 2008; Lippold et al., 2017; Kishore et al., 2011b).

The degradation of polysorbates by oxidation and chemical or enzymatic hydrolysis are well known and well studied. The latter mechanism is reported to be driven primarily by host cell proteins such as lysosomal phospholipase A2 (LPLA2), which co-purifies with the protein of interest and can catalyze the cleavage of ester bonds in polysorbates. (Labrenz, 2014; Dixit et al., 2016).

These enzymes were recently reported to have different specificities for monoesters or higher order species of polysorbate, resulting in different PS degradation patterns (Graf et al., 2020; Hall et al., 2016). Hydrolysis of PS20 not only results in loss of surfactant functionality (Kishore et al., 2011a), but also results in the release of free fatty acids (FFA), such as lauric acid or myristic acid, which are difficult to dissolve in aqueous solutions. It is soluble and can form visible or subvisible particles when the FFA concentration exceeds the solubility limit. The solubility of FFAs in solution depends on a variety of different factors including temperature, pH or the concentration of residual intact polysorbate (Doshi et al., 2015). Trace levels of metal ions such as Al ³⁺ interact with FFAs resulting from hydrolytic PS decomposition, ultimately resulting in FFA-metal complexes that precipitate out of the aqueous formulation and act as nucleation species for visible particles. has been previously shown (Allmendinger et al., 2021).

However, the occurrence of FFA particle formation in biopharmaceuticals is often unpredictable, leading to the assumption that other nucleation factors may be involved in particle formation. Thus, there remains a need to provide a solution that combats the formation of visible particles in parenteral aqueous protein formulations, eg aqueous preparations (or compositions) of antibodies.

The present invention provides a solution to this problem. More particularly, the present invention provides the option of mitigating FFA particle formation below the solubility limit through the addition of excipients (chelating agents), which complex polyvalent cations and fatty acids resulting from polysorbate degradation. can be prevented from interacting with

Chelating agents such as EDTA or DTPA have been commonly used in biopharmaceutical formulations to prevent oxidative degradation of proteins or polysorbates (Yarbrough et al., 2019; Doyle Drbohlav et al., 2019; Kranz et al., 2019; Doshi et al., 2021; Gopalrathnam et al., 2018). Oxidation may be facilitated by the presence of transition metals, which may originate from stainless steel production equipment (Zhou et al., 2011) or may be introduced through raw materials such as histidine (European Pharmaceutical Quality Council).

In one embodiment, the present invention provides a stabilizing agent comprising a protein and further comprising at least one chelating agent, together with pharmaceutically acceptable excipients such as buffers, stabilizing agents, including antioxidants. to provide an aqueous composition.

In one embodiment, the invention provides the use of chelating agents to prevent the formation of visible particles in aqueous protein formulations.

In one embodiment, the present invention provides the use of chelating agents in aqueous protein formulations to prevent the formation of visible particles containing concentrations of free fatty acids below solubility levels.

In another embodiment, the invention provides a pharmaceutical dosage form comprising a preparation, eg an aqueous antibody composition, as defined herein in a container or vial.

Hydrodynamic radius (rH) of laurate particles over time as a function of Al concentration. Inflection points of (A) DLS intensity and (B) sigmoidal fit of laurate particles over time as a function of Al concentration. (A) Hydrodynamic size and (B) scattering intensity of laurate particles over time as a function of metal cation type and concentration. Hydrodynamic particle radius of laurate particles over time in the presence of (A) EDTA and (B) DTPA (C) GLDA and (D) PDTA as a function of chelator to aluminum ratio. Scattering intensity of laurate particles over time in the presence of (A) EDTA and (B) DTPA (C) GLDA and (D) PDTA as a function of chelator to aluminum ratio. (A) Scattering intensity and (B) hydrodynamic particle radius of laurate particles over time as a function of DTPA to Fe ratio. (A) Scattering intensity and (B) hydrodynamic particle radius of laurate particles over time as a function of DTPA to Al ratio.

DETAILED DESCRIPTION OF THE INVENTION The formation of visible particles consisting of free fatty acids (FFA) as a result of surfactant degradation, in particular polysorbate (PS20 and/or PS80) degradation, is useful for parenteral preparations of eg therapeutic antibodies. The limited choice of surfactants in parenteral protein formulations is a major challenge in the biopharmaceutical industry. Reduce or even eliminate polysorbate degradation and release of FFAs by various means, as FFAs can precipitate to form visible particles, which in turn can affect parenteral drug quality. is important.

Commercially available polysorbates (PS20 and 80) are chemically diverse mixtures containing primarily sorbitan POE fatty acid esters. The main species of PS80 contains a sorbitan headgroup with four polyoxyethylene (POE) chains extending from the sorbitan headgroup. Theoretically, there are a total of 20 POE units associated with each headgroup, but in practice there may be more or less. Typically, the number of POE units has a Gaussian-like distribution, resulting in a heterogeneous mixture. Of the four POE groups attached to the sorbitan headgroup, one to three of them are esterified to fatty acids (FA) at their ends, which can be terminated with primary alcohols. The FAs found in PS80 are 14-18 carbons long and can have up to 3 double bonds along the chain. The most abundant FA is oleic acid (≧58%, 18 carbons, 1 double bond) followed by linoleic acid (18%, 18 carbons, 2 double bonds). The number of FA substitutions on an individual sorbitan headgroup can range from 0-4. PS80 also has an isosorbide head group with 0-2 FA substitutions. There is also a significant amount of POE-FAs that are not bound to headgroups. All of these components result in variably heterogeneous mixtures that can vary widely between manufacturers. (Journal of Pharmaceutical Sciences 109 (2020) 633-639). For example, other fatty acids present in PS20 include caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid.

PS20 and 80 are available in various grades. According to the invention the following grades were tested:
- High Purity (HP) PS20
- Ultra-refined (SR) PS20
- High Purity (HP) PS80
- Super-refined (SR) PS80
- pure oleic acid (POA) PS80

All grades were purchased from Croda (herein "Croda"), a specialty chemicals company headquartered in Snaith, England. Compared to the HP PS20 and HP PS80 synthesis processes, the SR grade is further purified by a unique flash chromatography process that can remove additional polar and oxidative impurities such as aldehydes and peroxides from the PS raw material ( Doshi et al., 2020a). A grade of pure oleic acid was recently introduced by the Chinese Pharmacopoeia (ChP) Commission, which stipulated that the content of oleic acid ester should be ≧98.0% for injectable products. Although this high oleic acid grade is no longer essential for parenteral product use, it has a lower tendency to form subvisible and visible particles upon hydrolysis compared to HP/SR PS20 and HP/SR PS80 (Doshi et al. 2021), which has recently garnered greater popularity.

(Table 1a) US/European/Chinese (Ch) Pharmacopoeia Specifications for PS20 and PS80

According to the present invention, we investigated the role of polyvalent cations as nucleation factors for the formation of visible particles, spiked with free fatty acid solutions and partially degraded polysorbate enzymatically hydrolyzed with different enzymes. This has been proven in research. Metallic impurities, namely aluminum, calcium, magnesium, iron, and zinc, are introduced into biopharmaceutical products through the manufacturing process (process leachables) (Zhou et al., 2011) or primary packaging containers (glass leachables) (Ditter et al., 2018). There is a possibility that Some of these metal impurities, such as iron, are known to promote the oxidative degradation of polysorbates (Kranz et al., 2019; Doyle Drbohlav et al., 2019).

Thus, in one embodiment, the invention comprises a protein, and further comprises at least one chelating agent, together with pharmaceutically acceptable excipients such as buffers, stabilizers, including antioxidants. , to provide stable aqueous compositions. In one embodiment, said stable aqueous composition (or preparation) is for parenteral use.

In another embodiment, the present invention comprises a protein, together with pharmaceutically acceptable excipients such as buffers, stabilizers, including antioxidants, and free fatty acids, inorganic metal ions, and at least one A stable aqueous composition is provided which further comprises a chelating agent of the type. In one embodiment, said stable aqueous composition (or preparation) is for parenteral use. In another embodiment, free fatty acids are as defined herein. In yet another embodiment, said free fatty acids result from hydrolysis of PS20 or PS80. In yet another embodiment, said free fatty acids are present in said stable aqueous composition at a concentration less than their dissolved concentration, and the concentration of chelating agent is at least the same as the concentration of inorganic metal ions (i.e., equimolar ). In this embodiment, the inorganic metal ions may be one or several ions selected from multivalent ions of aluminum, calcium, magnesium, iron and/or zinc, preferably aluminum or iron.

In one embodiment, the "chelating agent" is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA or pentetate), ethylene glycol-bis(β-aminoethyl)-N,N,N',N'- Tetraacetic acid (EGTA), N-carboxymethyl-N'-(2-hydroxyethyl)-N,N'-ethylenediglycine (HEDTA), ethylenediamine-N,N'-bis(2-dihydroxyphenylacetic acid) (EDDHA) ), 1,3-diaminopropane-N,N,N',N'-tetraacetic acid (PDTA), N,N-bis(carboxymethyl)-L-tetrasodium glutamate (GLDA), citrate, malonate, tartrate , ascorbate, salicylic acid, aspartic acid, aspartic acid, glutamic acid. In another embodiment, said chelating agent is ethylenediaminetetraacetic acid (EDTA). In another embodiment, the chelating agent is diethylenetriaminepentaacetic acid (DTPA or pentetic acid). In yet another embodiment, only one chelating agent is used.

In one embodiment, the chelating agent is present at a concentration of 0.0005-2.0% (w/v), or 0.001-0.1% (w/v). In another embodiment, when the chelating agent is EDTA, it is present in a relative amount of 0.005% (w/v). In another embodiment, when the chelator is DTPA, it is present in an amount of 0.05 mM. In yet another embodiment, the chelating agent is present in at least the same (ie equimolar) amount as the metal impurity or inorganic metal ion in the composition according to the invention.

In another embodiment there is provided a composition as defined above, wherein the pH of said composition is in the range of 5-7. In one aspect, the pH is about 5.5 or about 6.

In another embodiment, the invention provides a composition as defined herein before, wherein the protein is an antibody. In one aspect, the antibody is a monoclonal antibody. In another aspect, the antibody is a human or humanized monoclonal, mono- or bispecific antibody.

In yet another embodiment, an antibody according to the invention is an antibody with INN Pertuzumab. Pertuzumab is commercially available, eg, under the brand name PERJETA®. Pertuzumab is also disclosed, for example, in EP2238172B1. Thus, in another embodiment, "Pertuzumab (or "rhuMab 2C4") refers to an antibody comprising the variable light and variable heavy amino acid sequences of SEQ ID NOs: 3 and 4, respectively, as disclosed in EP2238172B1. When pertuzumab is an intact antibody, pertuzumab comprises the light and heavy chain amino acid sequences of SEQ ID NOS: 15 and 16, respectively, as disclosed in EP2238172B1.

In another embodiment, the invention provides a composition as defined hereinbefore, consisting of the following components: Formulation A: 10 mM His/HisHCl, pH 5.0, 10 mM methionine, 240 mM sucrose, 10 mg/mL API in 0.05% (w/v) PS20; Formulation B: 20 mM His, pH 6, 240 mM trehalose, 25 mg/mL API in 0.02% (w/v) PS20; Formulation C: 20 mM L-His 50 mg/mL API in /His acetate buffer pH 5.5, 220 mM sucrose, 10 mM L-methionine, 0.04% (w/v) PS20; Formulation D: 20 mM L-His/His acetate buffer pH 5.5, 130 mM Arginine hydrochloride, 10 mM L-methionine, 0.04% (w/v) 180 mg/mL API in PS20; Formulation E: 20 mM His/Asp pH 6.0, 150 mM Arginine, 40 mM Met, 0.05% (w/v) ) 175 mg/mL API in PS80. The term "API" as used herein means active pharmaceutical ingredient and is well known to those skilled in the art of pharmaceutical preparation. In one embodiment the API is a protein or antibody as defined herein.

In another embodiment, the present invention provides any of the compositions designated Formulations 01, 02, 03, 04 or 05 as specified in Example 5 (Table 7).

In another embodiment, the invention provides a composition comprising 30 mg/mL pertuzumab in 20 mM histidine acetate buffer (pH 6.0), 120 mM sucrose, 0.2 mg/mL HP PS20, 10 mM methionine and 0.05 mM DTPA. offer.

In another embodiment, the invention provides a composition comprising 30 mg/mL pertuzumab in 20 mM histidine acetate buffer pH 6.0, 120 mM sucrose, 0.2 mg/mL HP PS20, 10 mM methionine and 0.05 mM EDTA .

In another embodiment, the invention comprises 30 mg/mL pertuzumab in 20 mM histidine acetate buffer pH 6.0, 120 mM sucrose, 0.2 mg/mL pure oleic acid (POA) PS80, 10 mM methionine and 0.05 mM DTPA. A composition is provided.

In another embodiment, the invention comprises 30 mg/mL pertuzumab in 20 mM histidine acetate buffer pH 6.0, 120 mM sucrose, 0.2 mg/mL pure oleic acid (POA) PS80, 10 mM methionine and 0.05 mM EDTA A composition is provided.

In another embodiment, the present invention provides a chelating agent as defined herein for the manufacture of a medicament, in particular for the manufacture of a stable parenteral protein preparation, more particularly a parenteral antibody preparation. provide use. In one embodiment the parenteral preparation is an aqueous preparation. In another embodiment, the parenteral preparation is for subcutaneous (sc) application. In another embodiment the parenteral preparation is for intravenous (iv) administration.

In another embodiment, the present invention provides the use of a chelating agent as defined herein for preventing the formation of visible particles in parenteral protein preparations, especially antibody preparations. In one aspect, the parenteral preparation is an aqueous preparation. In another aspect, the parenteral preparation is for subcutaneous (sc) application. In another aspect, the parenteral preparation is for intravenous (iv) administration. In another aspect, the present invention provides the use of a chelating agent as defined herein for preventing the formation of visible particles comprising concentrations of free fatty acids below the solubility level in parenteral protein preparations. .

The term "parenteral" as used herein has its ordinary meaning. In one aspect, parenteral means for subcutaneous (sc) and/or intravenous injection.

The parenteral protein preparation is "stable" due to the presence of a chelating agent as defined herein. The term "stable" means that the preparation remains free; or essentially free; or substantially free of visible particles until the end of its approved shelf life. means. In one aspect, the preparation is stable for up to 30 months; or up to 24 months; or up to 18 months; or up to 12 months. The stability of parenteral protein preparations can be influenced by parameters well known to those skilled in the art such as, for example, light (UV irradiation), temperature and/or shaking. Thus, in one aspect, the term "stable" refers to the non-stable, e.g. Includes conditions normally recommended for storage of products containing oral protein preparations or antibody preparations. In one embodiment, the term "stable" includes a period of 30 months at a temperature between 2°C and 8°C and substantially protected from light.

The presence of visible particles can generally be detected using methods described in the European Pharmacopeia or US Pharmacopeia (Ph. Eur 10.0; Section 2.9.20; and USP 37-NF 32 <790>, First Supplement). In one embodiment of the present invention, the term "free" of visible particles utilizes a Seidenader V90-T instrument (Seidenader Maschinenbau GmbH, Markt Schwaben, DE) using the methods described in the accompanying working examples. , meaning that no visible particles can be detected in parenteral protein preparations. The term "essentially free of visible particles" utilizes a Seidenader V90-T instrument (Seidenader Maschinenbau GmbH, Markt Schwaben, DE), using the methods and conditions described in the accompanying working examples, It means that 1-5 visible particles can be detected in a parenteral protein preparation. The term “substantially free of visible particles” refers to black and white panels (herein “E /P box” or “E/P”) means that 0-4 visible particles can be detected.

The term "visible particle" means a particle containing one or several free fatty acids or a mixture of aggregated proteins and free fatty acids. In one aspect, visible particles have a particle size of at least 80 μm or at least 100 μm and can be seen, for example, as turbidity or precipitates in parenteral protein preparations. In one embodiment, the visible particles are formed by at least one polyvalent cation and free fatty acids cleaved from surfactants (such as PS20 and PS80) present in parenteral protein preparations. The term "polyvalent cation," as used herein, means one or more metallic impurities that are introduced into a parenteral protein preparation through the manufacturing process or primary packaging container. In one embodiment, such polyvalent cations are cations selected from aluminum, calcium, magnesium, iron, zinc. The term "fatty acid" as used herein has its ordinary meaning known to those skilled in the art of organic chemistry. In one embodiment, the term fatty acid refers to any fatty acid(s) present in PS20 or PS80 or cleaved from PS20 or PS80. In another embodiment, the term "fatty acid" means lauric acid, or myristic acid, or palmitic acid, or stearic acid, or oleic acid. According to the present invention, said fatty acid may be present in the aqueous protein preparation at a concentration below its solubility concentration (or "solubility level") and is visible together with polyvalent cations defined herein as nucleating factors. Particles can be formed. The soluble concentrations of fatty acids as defined herein are well known to those skilled in the art and can be found for example in (Doshi et al., 2015; Doshi et al., 2020b; Glucklich et al., 2020). In one embodiment, the term "below its dissolved concentration" means below its dissolved concentration in an aqueous solution or buffer of a fatty acid as defined herein at any temperature between 0°C and 30°C. do. In another embodiment, the term "below its dissolved concentration" means below its dissolved concentration in an aqueous solution or buffer of a fatty acid as defined herein at a temperature of 2-8°C. In yet another embodiment, the term "below its dissolved concentration" means below its dissolved concentration in an aqueous solution or buffer of a fatty acid as defined herein at a temperature of about 5°C.

Thus, in yet another embodiment, the present invention provides the herein defined for the manufacture of a medicament, in particular for the manufacture of an aqueous parenteral protein preparation, more particularly a parenteral antibody preparation. and for the entire approved shelf life, but at least up to 30 months, or up to 24 months; or up to 18 months; or up to 12 months, and such preparations free fatty acids resulting from the degradation of PS20 or PS80 and, optionally, free or substantially free of visible particles, including one or several polyvalent cations, under conditions recommended for storage of or essentially free of

In another embodiment, the invention provides a pharmaceutical dosage form comprising a protein preparation, eg an aqueous antibody preparation, as defined herein in a container, eg a vial or syringe.

In another embodiment, the invention provides a pharmaceutical dosage form comprising a protein preparation resulting from the use of a chelating agent as defined herein in a container such as a vial or syringe.

The term "excipient" refers to an ingredient, other than the active ingredient, in a pharmaceutical composition or preparation that is non-toxic to the subject. Excipients include, but are not limited to, buffers, stabilizers, including antioxidants, or preservatives.

The term "buffer" is well known to those skilled in the art of organic chemistry or pharmacy, eg, the development of pharmaceutical preparations. Buffers as used herein refer to acetate, succinate, citrate, arginine, histidine, phosphate, tris, glycine, aspartate, and glutamate buffer systems. Further, in this embodiment, the histidine concentration of said buffer is 5-50 mM. Preferred buffers are free histidine bases and histidine-HCl or acetate or succinate and/or aspertate. Further, in this embodiment, the histidine concentration of said buffer is 5-50 mM.

The term "stabilizer" is well known to those skilled in the art of organic chemistry or pharmacy, eg the development of pharmaceutical preparations. Stabilizers according to the present invention are selected from the group consisting of sugars, sugar alcohols, sugar derivatives or amino acids. In one aspect, the stabilizer is (1) sucrose, trehalose, cyclodextrin, sorbitol, mannitol, glycine, or/and (2) methionine, and/or (3) arginine, or lysine. In yet another aspect, the concentration of said stabilizer is (1) up to 500 mM, or (2) 5-25 mM, or/and (3) up to 350 mM, respectively.

The term "protein" as used herein means any therapeutically important polypeptide. In one embodiment, the term protein refers to an antibody. In another embodiment the term protein refers to an immunoconjugate.

The term "antibody" is used herein in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), as long as they exhibit the desired antigen-binding activity. and antibody fragments, including but not limited to various antibody classes or structures. In one embodiment, any of these antibodies are human or humanized. In one aspect, the antibody is alemtuzumab (LEMTRADA®), atezolizumab (TECENTRIQ®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®) ), pertuzumab (OMNITARG/PERJETA®, 2C4), trastuzumab (HERCEPTIN®), tositumomab (Bexxar®), abciximab (REOPRO®), adalimumab (HUMIRA®) , apolizumab, acelizumab, atolizumab, bapineuzumab, basiliximab (SIMULECT®), bavituximab, belimumab (BENLYSTA®), briankinumab, canakinumab (ILARIS®), cedelizumab, certolizumab pegol (CIMZIA®), cidofucizumab, sidutuzumab, cixutumumab, clazakizumab, crenezumab, daclizumab (ZENAPAX®), darotuzumab, denosumab (PROLIA®, XGEVA®), eculizumab (SOLIRIS®) )), efalizumab, epratuzumab, erulizumab, emicizumab (HEMLIBRA®), felubizumab, vontolizumab, golimumab (SIMPONI®), ipilimumab, imugatuzumab, infliximab (REMICADE®), labetuzumab, lebrikizumab, lexatumumab, lintuzumab, rucatumumab, rulizumab pegol, rumretuzumab, mapatumumab, matuzumab, mepolizumab, mogamulizumab, motavizumab, motovizumab, muronomab, natalizumab (TYSABRI®), necitumumab (PORTRAZZA®), nimotsumab Zumab (THERACIM®) , Norobizumabu, Numabizumab, Orokizumab, Omari Mab (XOLAIR (registered trademark)), onal -Tsumabu (known as METMAB), Paris Bizumab (Synagis (registered trademark)), Pascorizumab, Pasufushi Tsumabu, Pemblolizumab (Pembrisumabu KEYTRUDA (registered trademark), Pexerizumab, priliximab, ralivizumab, ranibizumab (LUCENTIS®), reslibizumab, reslizumab, lesivizumab, lovatumumab, lontarizumab, lobelizumab, ruplizumab, sarilumab, secukinumab, cerivantumab, cifalimumab, sibrotuzumab, siltuximab (SYLVANT ( registered trademark)), siplizumab, sontuzumab, selected from: taducizumab, talizumab, tefibazumab, tocilizumab (ACTEMRA®), tralizumab, tuxituzumab, umabizumab, ultoxazumab, ustekinumab (STELARA®), vedolizumab (ENTYVIO®), bicilizumab, zanolimumab, zalutumumab .

"Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single chain antibody molecules (eg scFv and scFab); single domains Antibodies (dAbs); and multispecific antibodies formed from antibody fragments, but are not limited to them. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chains. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which have subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, It can be subdivided into IgA1, and IgA2. In certain aspects, the antibody is of the IgG1 isotype. In certain aspects, the antibody is of the IgG1 isotype with P329G, L234A and L235A mutations to reduce Fc region effector function. In another aspect, the antibody is of the IgG2 isotype. In certain aspects, the antibody is of the IgG4 isotype with a S228P mutation in the hinge region to improve stability of the IgG4 antibody. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

A "human antibody" is an antibody that has an amino acid sequence corresponding to that of a human or produced by human cells, or derived from a human antibody repertoire or other human antibody coding sequences, and derived from non-human sources. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein all or substantially all of the CDRs correspond to those of a non-human antibody, and the FR correspond to FRs of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, eg, a non-human antibody, refers to an antibody that has undergone humanization.

The term "hypervariable region" or "HVR", as used herein, refers to regions of antibody variable domains that are hypervariable in sequence and determine antigen-binding specificity, e.g., "complementarity determining regions" ( CDR). In general, antibodies contain 6 CDRs, 3 in VH (CDR-H1, CDR-H2, CDR-H3) and 3 in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:
(a) occurs at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3); hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) occur at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) CDRs (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and (c) amino acid residues 27c-36 (L1), 4 6 to 55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732- 745 (1996)).

Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will appreciate that CDR designations may also be determined according to Chothia, supra, McCallum, supra, or any other scientifically acceptable nomenclature system.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "individual" or "subject" is a mammal. Mammals include domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and rats), including but not limited to. In certain aspects, the individual or subject is human.

An "isolated" antibody is one that is separated from a component of its natural environment. In some aspects, antibodies are determined, for example, by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reversed-phase HPLC) methods. , purified to greater than 95% or 99% purity. For a review of methods for assessment of antibody purity, see, eg, Flatman et al., J. Am. Chromatogr. B 848:79-87 (2007).

The term "pharmaceutical composition" or "pharmaceutical preparation" means that the active ingredients contained therein are in such a form as to render effective the biological activity of the active ingredients and to which the pharmaceutical composition will be administered. Refers to preparations that do not contain additional ingredients that are unacceptably toxic to the skin.

"Pharmaceutically acceptable carrier" refers to an ingredient, other than the active ingredient, in a pharmaceutical composition or preparation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, excipients as defined herein.

A. Chimeric and Humanized Antibodies In certain aspects, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (eg, from a mouse, rat, hamster, rabbit, or non-human primate, eg, monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain aspects, a chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from non-human antibodies and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally may also comprise at least a portion of a human constant region. In some aspects, some FR residues of the humanized antibody are modified from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity. Replaced by the corresponding residue.

Humanized antibodies and methods for their production are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), see, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. , Methods 36:25-34 (2005) (describing a specificity determining region (SDR) graft); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-. 68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (e.g., Sims et al., J. Immunol .151:2296 (1993)); framework regions derived from the consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (e.g., Carter et al., Proc. Natl. Acad. Sci. USA); 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (e.g., Baca et al., J. Biol. Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

B. Human Antibodies In certain aspects, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically have all or part of the human immunoglobulin loci replacing the endogenous immunoglobulin loci, or existing extrachromosomally or randomly integrated into the animal's chromosomes. include. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE™ technology; U.S. Pat. No. 5,770,429, which describes the HUMAB® technology. US Pat. No. 7,041,870 describing KM MOUSE® technology; and US Patent Application Publication No. 2007/0061900 describing VELOCIMOUSE® technology. Human variable regions from intact antibodies generated by such animals may be further modified, eg, by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human xenomyeloma cell lines have been described for the production of human monoclonal antibodies. (eg, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987). ); and Boerner et al., J. Immunol , 147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology have also been described by Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, US Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (human - describing human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185- 91 (2005).

Human antibodies can also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

C. Antibody Derivatives In certain aspects, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known and readily available in the art. Suitable sites for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3 ,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene Examples include, but are not limited to, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if multiple polymers are attached they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization is not limited, but the specific property or function of the antibody that is to be improved, the antibody derivative being of therapeutic use under the defined conditions. can be determined based on considerations including whether

D. Immunoconjugates The present invention also provides cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins; enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), Alternatively, immunoconjugates are provided comprising an antibody herein conjugated (chemically linked) to one or more therapeutic agents, such as radioisotopes.

In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC), in which the antibody is conjugated to one or more of the aforementioned therapeutic agents. Antibodies are typically attached to one or more therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents and drugs and linkers, is provided in Pharmacol Review 68:3-19 (2016).

In another aspect, the immunoconjugate comprises diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha - sarcin, Aleurites fordii protein, diancin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, As described herein conjugated to enzymatically active toxins or fragments thereof, including but not limited to saponaria officinalis inhibitors, gelonin, mitgerin, restrictocins, phenomycins, enomycins, and trichothecenes. containing antibodies of

In another aspect, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. Various radioactive isotopes are available for the production of radioconjugates. Examples include radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu. When used for detection, the radioconjugates are radioactive atoms such as tc99m or I123 for scintigraphic studies, or spins for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri). Labels (again, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron) can be included.

Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents: for example, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (dimethyladipimidate HCl, etc.), active esters (disuccinimidyl suberate etc.), aldehydes (glutaraldehyde, etc.), bis-azido compounds (bis (p-azidobenzoyl) hexanediamine, etc.), bis-diazonium derivatives (bis-(p-diazoniumbenzoyl)-ethylenediamine, etc.), diisocyanates (toluene 2, 6-diisocyanate, etc.), and bis-active fluorine compounds (1,5-difluoro-2,4-dinitrobenzene, etc.). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug within the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); US Pat. No. 5,208,020) have been used. may

Immunoconjugates or ADCs herein are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, Ill., USA), BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, but are not limited to , MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-( Conjugates prepared using cross-linker reagents including, but not limited to, 4-vinylsulfone)benzoate) are expressly contemplated.

E. Multispecific Antibodies In certain aspects, the antibodies provided herein are multispecific antibodies, eg, bispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has binding specificities for at least two different sites, ie, different epitopes on different antigens or different epitopes on the same antigen. In certain aspects, a multispecific antibody has more than two binding specificities. Multispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (Milstein and Cuello, Nature 305:537 (1983)). ) and "knob-in-hole" manipulations (see, eg, US Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies may also be engineered by electrostatic steering effects to create antibody Fc heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (e.g., US See Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); production of bispecific antibodies using leucine zippers (e.g., Kostelny et al., J. Immunol., 148 ( 5):1547-1553 (1992) and WO2011/034605); use of common light chain technology to avoid the light chain mispairing problem (e.g., WO98/ 50431); the use of "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)). ); and the use of single-chain Fv (sFv) dimers (see, eg, Gruber et al., J. Immunol., 152:5368 (1994)); Immunol. 147:60 (1991).

Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies," or DVD-Igs (see, eg, WO2001/77342 and WO2001/77342 and 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites are WO2010/115589, WO2010/112193, WO2010/136172, WO2010/145792 , and in WO2013/026831. Bispecific antibodies or antigen-binding fragments thereof also include "Dual Acting FAbs" or "DAFs" that contain antigen binding sites that bind to two different antigens or two different epitopes of the same antigen. (See, e.g., U.S. Patent Application Publication No. 2008/0069820 and International Publication No. WO2015/095539).

Multispecific antibodies may also be in asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e. VH/VL domains (e.g. WO2009/080252 and WO2015/ 150447), CH1/CL domains (see e.g. WO2009/080253) or complete Fab arms (see e.g. WO2009/080251, WO2016/016299, Schaefer et al., PNAS, 108 (2011) 1187-1191 and Klein et al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises cross-Fab fragments. The term "cross-Fab fragment" or "xFab fragment" or "cross-over Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. Cross-Fab fragments are composed of a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a poly chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Contains peptide chains. Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, for example, WO2016/172485.

Various additional molecular formats for multispecific antibodies are known in the art and included herein (see, eg, Spiess et al., Mol Immunol 67 (2015) 95-106).

F. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

For native antibodies or native antibody fragments where two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Such nucleic acids encode amino acid sequences comprising the VL and/or VH of the antibody (eg, the light and/or heavy chain(s) of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

For bispecific antibodies with heterodimeric heavy chains, four nucleic acids are required, one for the first light chain and one for the first comprising the first heteromonomeric Fc region polypeptide. one for the second light chain and one for the second heavy chain comprising the second heteromonomeric Fc region polypeptide. The four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. Such a nucleic acid may be an amino acid sequence comprising the first VL and/or an amino acid sequence comprising the first VH comprising the first heteromonomeric Fc region and/or an amino acid sequence comprising the second VL of the antibody or encodes a second VH-comprising amino acid sequence (e.g., first and/or second light chain and/or first and/or second heavy chain of an antibody) comprising a second heteromonomeric Fc region . These nucleic acids can be on the same expression vector or on different expression vectors, usually these nucleic acids are located on two or three expression vectors, i.e. one vector contains these nucleic acids can include two or more of Examples of these bispecific antibodies are Cross Mabs (see, eg, Schaefer, W. et al., PNAS, 108 (2011) 11187-1191). For example, according to the EU index numbering, one of the heteromonomer heavy chains contains a so-called "knob mutation" (T366W and optionally one of S354C or Y349C) and the other a so-called "hole mutation" (T366S, L368A and Y407V, and optionally Y349C or S354C) (see, eg, Carter, P. et al., Immunotechnol. 2 (1996) 73).

For recombinant production of antibodies, for example, nucleic acids encoding the antibodies described above are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody). ) sequenced or produced by recombinant methods or obtained by chemical synthesis.

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. See, eg, US Pat. No. 5,648,237, US Pat. No. 5,789,199, and US Pat. No. 5,840,523 for expression of antibody fragments and polypeptides in bacteria. (Charlton, K. A. Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (eds.), Humana Press, Totowa, describing the expression of antibody fragments in E. coli. NJ (2003), pp. 245-254) After expression, the antibody may be isolated from the bacterial cell paste in the soluble fraction and further purified.

In addition to prokaryotes, eukaryotes such as filamentous fungi and yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast that have been "humanized" in their glycosylation pathways. Strains are included that result in the production of antibodies with partially or fully human glycosylation patterns. Gerngross, T.; U.S.A. , Nat. Biotech. 22 (2004) 1409-1414; and Li, H.; et al., Nat. Biotech. 24 (2006) 210-215.

Also suitable host cells for the expression of the (glycosylated) antibody are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified and may be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. For example, US Pat. No. 5,959,177, US Pat. No. 6,040,498, US Pat. No. 6,420,548, US Pat. No. 7,125,978 and US Pat. (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 strain (COS-7); human embryonic kidney strains (eg Graham, FL et al., J. Gen Virol. (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (eg, Mather, JP, Biol. Reprod. 23 (1980) 243). monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells ( human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); ) 44-68), MRC5 cells, and FS4 cells Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells (Urlaub, 2000), including DHFR-CHO cells. USA 77 (1980) 4216-4220), and myeloma cell lines such as Y0, NS0 and Sp2/0. For reviews of animal host cells, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, B.K.C. 2004), pp. 255-268.

The invention will now be further illustrated by the following non-limiting working examples.

Materials and Methods Free Fatty Acid Solubility in Buffered Aqueous Solutions Free fatty acid stock solutions were prepared as previously described by Doshi et al. (Doshi et al., 2015) with minor modifications. Briefly, lauric acid (“LA”, Sigma-Aldrich/Merck, Darmstadt, DE) and myristic acid (“MA”, Sigma-Aldrich/Merck, Darmstadt, DE) were added to PS20 HP (Croda, Edison, NJ, USA) and stirred (150 rpm) at 60° C. for 30 minutes until both FFAs were completely dissolved. The solution was diluted 1:5 with preheated (60° C.) water for injection (WFI) and immediately filtered through a 0.22 μm PVDF Steriflip filter (Merck Millipore, Darmstadt, DE). The concentrations of LA and MA in the FFA stock solutions were verified by LC-MS as described by Honemann et al. (Honemann et al., 2019).

The LA/MA/PS20 stock solution was spiked (diluted 1:500, n=3) into 20 mM histidine acetate buffer pH 5.5 and washed in a MaxQ™ 4000 Benchtop Orbital Shaker (Thermo Scientific™, Waltham, MA, USA). ) at 25° C. for 1 hour. Samples were stored at 5°C and after 0, 1, 7 and 28 days Ph. Eur. A black/white panel according to 2.9.20 (European Pharmaceutical Quality Council) and a Seidenader V90-T instrument (Seidenader Maschinenbau GmbH, Markt Schwaben, Del.) were used to analyze for visible particles. All samples were visually inspected after equilibration to ambient temperature (1 hour). The number of visible particles per container is defined as “many particles (>7)”, “few particles (5-7)”, or “substantially free of particles (0-4)” in the E/P box. ” and defined by Seidenader as “many particles (>10),” “few particles (6-10),” “essentially particle-free (1-5),” or “particle-free (0)”.

Compositions of FFA stock solutions and samples are outlined in Table 1.

(Table 1) Composition of LA/MA/PS20 stock solutions and samples

Spike Studies with Free Fatty Acids and Aluminum A 100 ppm aluminum stock solution was prepared from aluminum chloride hexahydrate in 20 mM histidine acetate pH 5.5. The actual concentration of aluminum was determined by inductively coupled plasma mass spectrometry (ICP-MS). This stock solution was then diluted to 10 ppm Al ³⁺ and - sterile filtered through a 0.22 μm porosity filter cartridge (Sterivex-GV, Millipore). Further dilutions (10-250 ppb Al ³⁺ ) were prepared aseptically under laminar airflow and dispensed into 20 mL type I borosilicate glass vials (Schott, Mainz, Del.).

Samples containing different amounts of aluminum (0-250 ppb) were spiked with different FFA stock solutions (LAMA-2, LAMA-6, LAMA-7, LAMA-8 and LAMA-10). Samples containing 250 ppb aluminum were further spiked with ethylenediaminetetraacetic acid (EDTA) to give a target concentration of 0.005% (w/v). All vials were sealed with 20 mm Teflon coated injection stoppers (D777-1, Daikyo) and aluminum crimp caps and placed in a MaxQ™ 4000 Benchtop Orbital Shaker (Thermo Scientific™, Waltham, Mass., USA) at 25°C. homogenized for 1 hour.

Dilutions spiked with LAMA-6, LAMA-7 and LAMA-8 yielded LA and MA concentrations below their solubility limits, whereas spikes with LAMA-2 yielded FFA concentrations above their solubility limits, Used as a positive control. LAMA-10 contained polysorbate 20 only and was used to prepare the negative control. All samples were prepared in triplicate.

Samples were stored at 5° C. and visible particle formation was assessed for up to 28 days using a black/white and Seidenader V90-T apparatus (Seidenader Maschinenbau GmbH, Markt Schwaben, Del.) as described above.

Solubility of partially degraded PS20. • Enzymatically hydrolyzed with candida antarctica lipase (CAL). A set of polysorbates with 6 different degradation levels (10%, 15%, 20%, 30%, 40% and 60% degradation) was prepared for each enzyme (Table 2).

A PS20 stock solution (50 mg/mL) was spiked (diluted 1:125, n=3) into 20 mM histidine buffer pH 5.5 and shaken in a MaxQ™ 4000 Benchtop Orbital Shaker (Thermo Scientific™, Waltham, Mass., USA). ) at 25° C. for 1 hour. Samples were stored at 5°C and after 0, 1, 7 and 28 days Ph. Eur. A black/white panel according to 2.9.20 (European Pharmaceutical Quality Council) was used to analyze for visible particles. All samples were visually inspected after equilibration to ambient temperature (1 hour). The number of visible particles per container was defined as "many particles (>7)", "few particles (5-7)", or "substantially free of particles (0-4)".

(Table 2) Enzymatically degraded polysorbates

Spike Studies with Partially Degraded Polysorbate 20 and Aluminum Diluted aluminum solutions in 20 mM histidine acetate pH 5.5 were prepared as above and added to 20 mL type I borosilicate glass vials (Schott, Mainz, DE). Samples containing different amounts of aluminum (0-250 ppb) were spiked with different PS20 stock solutions (PS20-Std, MML-10, MML-15, MML-40, CAL-10, CAL-15). Additionally, samples containing 250 ppb aluminum were supplemented with 0.005% (w/v) ethylenediaminetetraacetic acid (EDTA) or 0.05mM diethylenetriaminepentaacetic acid (DTPA) followed by partially degraded PS20. spiked. Vials were sealed with 20 mm Teflon coated injection stoppers (D777-1, Daikyo) and aluminum crimp caps and shaken in a MaxQ™ 4000 Benchtop Orbital Shaker (Thermo Scientific™, Waltham, Mass., USA) at 25° C. for 1 Time homogenized.

Samples prepared with MML-10/-15 and CAL-10/-15 yielded FFA concentrations below their solubility limit, whereas spikes with MML-40 (positive control) exceeded the solubility limit. resulting in greater FFA concentrations. PS20-Std contains only undegraded polysorbate 20 and was used to prepare the negative control. All samples were prepared in triplicate.

Samples were stored at 5°C and Ph. Eur. Particle formation was assessed up to 28 days by visual inspection using a black/white panel according to 2.9.20 (European Pharmaceutical Quality Council).

Example 1: Solubility of Free Fatty Acids in Histidine Acetate Buffer pH 5.5 Free fatty acid (FFA) stock solutions of lauric acid (LA) and myristic acid (MA) were spiked into 20 mM histidine acetate buffer pH 5.5 ( n=3). Samples were analyzed for the presence of visible particles using (A) Seidenader and (B) E/P boxes after incubation at 2-8° C. for 0, 1, 7 and 28 days. The number of particles per container is defined in the E/P box as “many particles (>7, xxx)”, “few particles (5-7, xx)”, or “substantially free of particles (0 ~4,/)”, and by Seidenader, “many particles (>10, xxx)”, “few particles (6-10, xx)”, “essentially particle-free (1-5, x )”, or “particle free (0,/)”. d=day of examination, d0=post-spike. Results are summarized in Table 3.

(Table 3) Solubility of free fatty acids in histidine acetate buffer pH 5.5

Example 2 Formation of Visible Particles After spiking the FFA stock solution into a buffered aqueous solution (20 mM histidine acetate buffer pH 5.5) containing different amounts of aluminum ranging from 0 to 250 ppb (n=3). Samples containing the highest amount of aluminum (250 ppb) were further formulated with a chelating agent (EDTA). Samples were stored at 2-8° C. for 0, 1, 7 and 28 days and then analyzed for the presence of visible particles using (A) Seidenader and (B) E/P boxes. Cumulative content of particles per container is defined as “majority of particles (>7, xxx)”, “minority of particles (5-7, xx)”, or “substantially free of particles” in the E/P box. (0-4,/)”, and by Seidenader, “many particles (>10, xxx)”, “few particles (6-10, xx)”, “essentially particle-free (1-5 , x)”, or “particle free (0,/)”. Samples containing no FFAs or no salt were used as negative controls, while samples containing FFAs above the solubility limit (*) were used as positive controls. d = day of examination. d0 = after spike. nd=undecided. Results are summarized in Table 4.

(Table 4) Formation of visible particles

Example 3: Solubility of partially degraded polysorbate 20 in histidine acetate buffer pH 5.5 Polysorbate 20 (PS20) pre-degraded by MML or CAL (degradation level 0-60%) was added to 20 mM histidine acetate. Buffer pH 5.5 was spiked (n=3) to a final PS20 concentration of 0.04% (w/v). Samples were visually inspected (E/P box) for the presence of visible particles after storage at 2-8° C. for 0, 1, 7 and 28 days. Cumulative content of particles per container is defined as “majority of particles (>7, xxx)”, “minority of particles (5-7, xx)”, or “substantially free of particles” in the E/P box. (0-4, /)”. d = day of examination. d0 = post spike, MML = Mucor miehei lipase, CAL = Candida antarctica lipase. Results are summarized in Table 5.

Table 5. Solubility of partially degraded polysorbate 20 in histidine acetate buffer pH 5.5

Example 4: Formation of Visible Particles After spiking partially degraded polysorbate 20 into a buffered aqueous solution (20 mM histidine acetate buffer pH 5.5) containing different amounts of aluminum ranging from 0 to 250 ppb (n=3) . Samples containing the highest amount of aluminum (250 ppb) were further formulated with a chelating agent (EDTA or DTPA). Samples were visually inspected (E/P box) for the presence of visible particles after 0, 1, 7 and 28 days of incubation at 2-8°C. The cumulative content of particles per container is defined as "majority of particles (>7, xxx)", "few particles (5-7, xx)", or "substantially free of particles (0-4,/ )”. Samples containing 100% intact PS20 or no salt were used as negative controls, while samples containing 60% degraded PS (MML) above the solubility limit (*) were used as positive controls. d = day of examination. d0 = post spike, nd = undetermined, MML = Mucor miehei lipase, CAL = Candida antarctica lipase. Results are summarized in Table 6.

Table 6 Formation of Visible Particles

Results The solubility limits of lauric acid and myristic acid were determined by spiking FFA stock solutions into histidine acetate buffer pH 5.5 to obtain target concentrations of 0-30 μg/mL for lauric acid and 0-12 μg/mL for myristic acid. (see Example 1). Samples were incubated at 2-8° C. and examined for visible particles using a Seidenader (Table 3A) and E/P box (Table 3B) after 0, 7, 14 and 28 days. Formation of numerous visible particles (>10 for Seidenader, >7 for E/P boxes) was already observed after 1 day in samples containing at least 20/8 μg/mL lauric/myristic acid, whereas the lower FFA Concentration resulted in overall lower particle counts and delayed particle development. Visual inspection by the Seidenader machine was performed at 1.5x magnification, so the total number of particles per vessel was higher compared to the E/P box analysis. The solubility limit was defined as the concentration of lauric acid and myristic acid above which many visible particles (>10 for the Seidenader and >10 for the E/P box were observed after 28 days for all three vials in triplicate sets). 7) was observed. As shown in Table 3, in samples containing at least 12.5 μg/mL lauric acid and 5 μg/mL myristic acid, free fatty acid concentrations exceeded the solubility limit and FFAs crashed out as visible particles. In conclusion, all samples with lower lauric and myristic acid concentrations were considered to be below the solubility limit.

The FFA stock solution was subsequently spiked into an aqueous buffer solution (20 mM histidine acetate buffer pH 5.5) containing different amounts of aluminum ranging from 0 to 250 ppb (Example 2). Thereby, the final concentrations of fatty acids in the samples were below the previously determined solubility limits (10/4, 7.0 μg/mL), except for one sample containing 25/10 μg/mL lauric/myristic acid, which was used as a positive control. 5/3, 5/2, 0/0 μg/mL lauric/myristic acid). Samples containing the highest content of aluminum (250 ppb) were additionally spiked with 0.005% (w/v) EDTA. All samples were incubated at 2-8° C. for up to 28 days and inspected for visible particles using the Seidenader (Table 4A) and E/P box (Table 4B). As shown in Table 4, the presence of aluminum resulted in the formation of FFA particles even below the solubility limit of FFA. The extent and occurrence of visible particle formation was dependent on aluminum concentration and surprisingly also inversely on FFA concentration. Samples with the highest concentration of aluminum showed the fastest particle generation and the highest number of particles. This effect was even more pronounced in samples containing reduced amounts of FFA. As demonstrated by the Seidenader results, even as little as 10 ppb of aluminum was sufficient to complex free fatty acids and form visible particles. Samples containing only fatty acids (below the solubility limit) but no aluminum or vice versa showed a significant number of visible particles (≤10 for Seidenader and E/ did not form ≦7) in the P-box.

The formation of visible FFA particles could be suppressed by adding 0.005% (w/v) EDTA to samples containing the highest aluminum concentration (250 ppb) and FFA levels below the solubility limit.

Turning to the more specific issue of FFA release from polysorbate in aqueous (parenteral) protein or antibody formulations, the hydrolysis of polysorbate co-purifies with the active pharmaceutical ingredient (API) to form an ester in polysorbate. It has been previously disclosed to be primarily driven by the presence of host cell proteins (HCPs) that can catalyze bond hydrolysis (Labrenz, 2014; Dixit et al., 2016). It is already known that different enzymes have different specificities for the monoester or polyester building blocks of polysorbate, resulting in different polysorbate degradation patterns (McShan et al., 2016).

In Example 3, polysorbate was artificially degraded by two different enzymes pre-immobilized on beads to allow precise control of degradation levels. MML preferentially degrades higher esters, whereas CAL uniformly degrades mono- and higher esters (Graf et al., 2020).

The solubility limits of partially hydrolyzed polysorbates degraded with either MML or CAL were determined by spiking PS20 stock solutions into histidine acetate buffer pH 5.5 to a total concentration of 0.4 mg/mL. Samples were incubated at 2-8° C. and visually inspected (E/P box) for the presence of visible particles up to 28 days. Formation of numerous visible particles (>7 in E/P boxes) was observed immediately after spiking with 40% and 60% degraded polysorbate by MML. In contrast, at the early time point (d0) no visible particles were observed for the CAL samples even at the highest degradation level (60%). After incubation at 2-8° C., visible particles were already observed at 20% or 30% polysorbate degradation for MML and CAL samples, respectively.

The solubility limit for each polysorbate degradation series was defined as the critical resolution above which many visible particles (>7 for the E/P box) were observed after 28 days for all three vials of the triplicate set. bottom.

Samples with lower polysorbate degradation (<20% for MML and <30% for CAL) were defined as below the solubility limit.

In the next step, the partially degraded PS20 solution was spiked into an aqueous buffer solution (20 mM histidine acetate buffer pH 5.5) containing different amounts of aluminum ranging from 0 to 250 ppb (Example 4). Thereby, the final concentrations of polysorbate degradants were below the previously determined solubility limits (10% and 15%), except for one sample containing 40% degraded polysorbate in MML, which was used as a positive control. rice field. Samples containing the highest content of aluminum (250 ppb) were further spiked with either 0.05 mM DTPA or 0.005% (w/v) EDTA. All samples were incubated at 2-8° C. for up to 28 days and visually inspected (E/P box). As shown in Table 6, the presence of aluminum resulted in the formation of FFA particles even below the critical polysorbate decomposition level. Again, both the extent of visible particle formation and particle generation depended on the aluminum concentration and surprisingly also inversely on the degree and pattern of degradation of PS20. Samples with the highest concentration of aluminum showed the fastest particle generation and the highest number of particles. This effect was slightly more pronounced at lower polysorbate degradation (10%). Interestingly, when aluminum was present, the CAL samples showed visible particle formation even at very low levels of aluminum (10 ppb), significantly earlier than the corresponding MML samples (after 7 days). .

No visible particles were formed in samples spiked with undegraded polysorbate or without aluminum when incubated at 2-8° C. for up to 28 days.

Thus, the formation of visible FFA particles was suppressed by adding 0.05 mM DTPA or 0.005% (w/v) EDTA to samples containing the highest aluminum concentration (250 ppb) and partially degraded polysorbate below the solubility limit. was successfully prevented by adding

Example 5: Particle formation in pertuzumab formulations with and without chelating agents Formulated at 30 mg/mL in 20 mM histidine acetate buffer pH 6.0, 120 mM sucrose, supplemented with 10 mM methionine and 0 or 0.05 mM chelating agent (DTPA or EDTA). The formulated drug product was filled into 20cc borosilicate vials (14.0 mL) and stored at 2-8°C. 30 vials of each formulation were prepared.

Table 7. Sample compositions of different mAb1 formulations

Visual inspection (Seidenader or E/P) was performed on 30 vials per formulation after 6 months of incubation at 2-8°C. Vials were inspected within 1 hour after removal from 2-8° C. storage (cold sample solution) or after equilibrating to ambient temperature for 4 hours.

result:
Tables 8 and 9 summarize visual inspection results using the E/P method or Sidenader, respectively, after 6 months of storage at 2-8°C. The overall particle count of the cold sample solution was generally higher compared to the sample after equilibration to ambient temperature, indicating that the particles were mainly free fatty acids or fatty acids with lower solubility at lower temperatures. It shows that it consists of salt. When comparing particles in different formulations after equilibration, the number of vessels containing particles was very similar for all five formulations (1-3 vessels out of 30). total number of particles and

(Table 8) Visual examination (EP) after 6 months (2-8°C)

(Table 9) Enhanced visual examination (Seidenader) after 6 months (2-8°C)

Example 6: Spike Studies Using Different PS20 and PS80 Grades and DTPA Methods Preparation of Enzymatically Degraded PS20 and PS80 Superpurified (SR) PS20 and PS80, High Purity (HP) PS20 and PS80, and Pure Oleic Acid (POA)PS80 was synthesized using immobilized enzymes (Graf et al., 2020) (Mukor miehei lipase (MML), Candida antarctica lipase (CAL) and Candida antarctica lipase B (CALB)). was enzymatically hydrolyzed to 10%. These enzymes have different specificities for mono- and higher esters, but MML mainly degrades higher ester species in PS, CAL targets mono- and higher esters, and CALB preferentially decomposes the monoester.

Spike Studies with Partially Degraded Polysorbates (PS20 and PS80) and Aluminum Diluted aluminum solutions in 20 mM histidine acetate pH 5.5 were prepared as above and added to 20 mL type I borosilicate glass vials ( Schott, Mainz, DE). Samples containing 0 or 250 ppb aluminum (Al ³⁺ ) were spiked with different PS stock solutions (HP PS20, SR PS20, HP PS80, SR PS80, POA PS80; degradation levels of 0 or 10%). Additionally, samples containing 250 ppb aluminum were spiked with partially degraded PS after supplementation with 0.05 mM diethylenetriaminepentaacetic acid (DTPA). Vials were sealed with 20 mm Teflon coated injection stoppers (D777-1, Daikyo) and aluminum crimp caps and shaken in a MaxQ™ 4000 Benchtop Orbital Shaker (Thermo Scientific™, Waltham, Mass., USA) at 25° C. for 1 Time homogenized.

Samples prepared with partially degraded PS20 or PS80 yielded FFA concentrations below the solubility limit. Different grades of undegraded polysorbate 20 and 80 were used as negative controls. All samples were prepared in triplicate.

Samples were stored at 5°C and subjected to Ph. Eur. 2.9.20. Particle formation was assessed up to 22 days by visual inspection using a black/white panel according to , and by enhanced visual inspection (Seidenader V90-T instrument).

Results Different grades of PS20 (SR, HP) and PS80 (SR, HP, POA) were partially degraded by 10% using different enzymes with different substrate specificities. PS stock solutions (0 or 10% degraded) were spiked into aqueous buffer solutions further containing either free (0 ppb) or 250 ppb aluminum, or 250 ppb aluminum and 50 μM DTPA. Vials were incubated at 2-8° C. and the formation of visible particles was monitored by visual inspection (E/P) and enhanced visual inspection (Seidenader). As shown in Table 10 (Visible Particles by E/P), no visible particles were observed during the course of the study for samples containing 10% degraded PS20 or PS80 but no aluminum. Many visible particles were formed in samples containing partially degraded PS20 and aluminum, regardless of the enzyme used for degradation, whereas for samples containing partially degraded PS80 and aluminum, visible particles were significantly less or not observed. No particles were observed in the presence of the chelating agent (DTPA) for all grades of PS. Interestingly, the undegraded HP PS20 and HP PS80 controls containing 250 ppb aluminum formed visible particles, whereas all other controls contained no particles. This observation may be explained by the fact that HP grade PS contains significantly higher levels of free fatty acids in the feed compared to the corresponding SR grade (Doshi et al., 2020a). These levels are well below FFA solubility levels, but may be high enough for FFA metal nucleation.

Table 10. Formation of Visible Particles After Spiking PS Stock Solution (0 or 10% Degradation Level) into Buffered Aqueous Solution (20 mM Histidine Acetate Buffer pH 5.5) Without Aluminum or with 250 ppb Aluminum (E/P) (n=3)
A sample containing 250 ppb aluminum was further formulated with a chelating agent (DTPA). Samples were stored for 0-22 days at 2-8° C. and then analyzed for the presence of visible particles using an E/P box. Cumulative content of particles per container (20 mL) may be "many particles (>7, xxx)", "few particles (5-7, xx)", or "substantially free of particles (0- 4, /)”. A sample containing any undegraded PS unsalted was used as a negative control. Results are reported as the average number of particles in the three vessels. d = day of examination, CALB = Candida antarctica lipase B, MML = Mucor miehei lipase, CAL = Candida antarctica lipase, DTPA = diethylenetriaminepentaacetic acid.

Table 11 shows the corresponding enhanced visual test results for HP PS20, SR PS20, HP PS80, SR PS80 and POA PS80. Controls containing partially decomposed PS in the absence of aluminum remained particle-free or essentially free, whereas all samples containing partially decomposed PS and aluminum , instantly formed many visible particles. Particle formation was significantly reduced in the presence of DTPA, regardless of the grade of PS or the enzyme used to degrade it. Again, many particles were formed in the undegraded HP PS20 and HP PS80 and samples containing aluminum, whereas few or no particles were observed in the other undegraded controls (with aluminum). .

Table 11 Formation of Visible Particles after Spiking PS Stock Solution (0 or 10% Degradation Level) into Buffered Aqueous Solution (20 mM Histidine Acetate Buffer pH 5.5) Without or with 250 ppb Aluminum (Seidenader) (n=3)
A sample containing 250 ppb aluminum was further formulated with a chelating agent (DTPA). Samples were stored at 2-8° C. for 0-22 days and then analyzed for the presence of visible particles using a Seidenader instrument. Cumulative content of particles per container: "Majority of particles (>10, xxx)", "Few particles (6-10, xx)", "Essentially free of particles (1-5, x) or "no particles (0,/). Samples containing undegraded PS or no salt were used as negative controls. d = day of testing, CALB = Candida antarctica. Lipase B, MML = Mucor miehei lipase, CAL = Candida antarctica lipase, DTPA = diethylenetriaminepentaacetic acid.

Example 7 Screening of Metal Salts and Chelating Agents Methods FFA Particle Nucleation by Dynamic Light Scattering (DLS) DLS experiments were performed on a DynaPro® plate reader (Wyatt, Santa Barbara, Calif.). The DLS plate reader is flushed with nitrogen 5 hours before starting the measurement and cooled to 5° C. for the entire duration of the measurement. 20 μg of lauric acid (LA) and various amounts of metal ions (Al ³⁺ , Fe ³⁺ , Zn ²⁺ , Mg ²⁺ , Ca ²⁺ ) in 20 mM L-histidine buffer at pH 6.0 supplemented with 6% v/v DMSO , Ni ²⁺ ) were mixed in a cuvette of a black glass bottom 96-well plate (Greiner Bio-One GmbH, Frickenhausen, Germany). FFA nucleation and particle growth were measured over 40-70 hours using a 633 nm laser and a backscatter detection system at 158°. The hydrodynamic particle radius (rH) was determined by fitting a cumulant fit to the resulting autocorrelation function. The lower and upper bounds of the cumulant fit were set to delay times τ of 10 μs and 1000 μs, respectively. The laser power was adjusted before each measurement sequence and kept constant throughout the measurement. Attenuation level was set to 0 for all measurements to collect the maximum amount of scattered light.

Effect of Aluminum (Al) Concentration on Particle Kinetics Al sample solutions (100×) in 20 mM histidine buffer pH 6.0 were prepared from sterile 50 ppm Al stock solution (20 mM glycine pH 2.5), 0, 20, 40, 60 and spiked into the DLS assay buffer at a target concentration of Al of 100 ppb. Grain nucleation and growth were measured over a period of 40 hours as described above.

式中、Ａは初期値を表し、Ｂは最終値を表し、ｘ_０はシグモイド曲線の中心または変曲点であり、ｄｘは時定数である。 Quantitative analysis was performed by analyzing the scattered laser light intensity of the FFA particles over time by fitting a sigmoidal Boltzmann function to the growth curve.

where A represents the initial value, B represents the final value, _x0 is the center or inflection point of the sigmoidal curve, and dx is the time constant.

Interaction of divalent and trivalent cations with lauric acid Aluminum (Al), iron (Fe), zinc (Zn), magnesium (Mg), calcium (Ca) and nickel (Ni) stock solutions were treated with at 4 mM in Milli-Q water pH 2.5 and stored at 2-8° C. until use. Diluted sample solutions (100×) were freshly prepared in Milli-Q water pH 2.5. DLS assay buffer was added to the salt sample solution to obtain metal ion target concentrations of 0, 1, 3, 10 and 30 μM. Particle size (rH) and strength were measured over a period of 70 hours as described above.

FFA metal nucleation in the presence of chelating agents. N-bis(carboxymethyl)-L-glutamate tetrasodium (GLDA) stock solutions were prepared in 20 mM histidine buffer pH 5.5 and diluted to desired concentrations in DLS assay buffer. For Al nucleation studies, a metal ion target concentration of 2 μM was used. The final concentrations of DTPA, EDTA, GLDA and PDTA were either 0, 0.5, 1.0, 1.5, 2 and 20 μM and the molar ratio of chelator to Al was 0, 0.25 and 0, respectively. .5, 0.75, 1.0 and 10. For Fe nucleation studies, the target concentration of Fe was 4 μM. The final concentration of DTPA was either 0, 0.04, 0.4, 2.0, 4.0 or 40 μM, respectively 0, 0.01, 0.1, 0.5, 1.0 and Resulting in a molar ratio (DTPA:Fe) of 10. DLS measurements were performed at 5° C. over 50 hours as described above.

DTPA Prevents FFA Metal Nucleation in the Presence of Actual Glass Leachates Representative glass leachates were prepared according to the procedure disclosed in Allmendinger et al. (Allmendinger et al., 2021). Briefly, 6 mL of pH 10 glycine solution was filled into 6 mL Fiolax® vials (Schott AG, Mullheim, Germany, and Schott North America Inc., NY, USA) and D777-1 serum stopper ( DAIKYO Seiko Ltd., Tokyo, Japan) and subjected to one autoclaving cycle (121° C., 20 minutes). The content of glass leachables in the sample after dilution was 37 ppb Al, 43 ppb B, 430 ppb Si, and 0 ppb Na, K, Ca, with 1.4 μM Al, 4.0 μM B and 15 0.3 μM Si. DTPA concentrations were either 0, 0.04, 0.4, 2.0, 4.0 or 40 μM and were 0, 0.03, 0.3, 1.5, 2.9 and 29, respectively. (DTPA:Al), or 0, 0.002, 0.02, 0.1, 0.2 and 1.9 (DTPA to glass leachate). DLS measurements were performed at 5° C. over 50 hours as described above.

Results Interaction of metal cations with lauric acid A surrogate assay based on DLS was established to assess the risk of other divalent and trivalent metal impurities that could potentially be introduced during DP manufacturing or storage. bottom. DLS can be used to capture particle formation and growth in the 0.3 nm-10 μm size range (Panchal et al., 2014), thus detecting early nucleation events (FFA-metal interactions) was suitable for In aqueous solution containing lauric acid (below the solubility limit), the major degradation product from hydrolyzed PS20, and DMSO (6% v/v), which increases LA solubility, since micelles of proteins and polysorbates can interfere with the assay. I made a measurement. In the first experiment, different Al concentrations were used to induce FFA complexation and subsequent particle formation. As shown in Figure 1, nanoparticles were formed in all formulations containing Al, whereas no particles were observed in controls containing neither Al nor FFA (data not shown). The grain size of the Al-containing samples increased over time, whereby the size increase increased at higher Al concentrations.

As shown in FIG. 2B, the scattering intensity of the Al-containing sample was initially higher compared to the Al-free control sample (5000 kCnt/s). A slight increase in strength over time was observed for the samples containing 40 ppb Al, while the samples containing 60-100 ppb Al showed a sharp increase in strength, which was fitted with a sigmoidal curve to give an inflection point. could be determined (Fig. 2B). It was not possible to use t _onset to assess grain growth kinetics, as the intensity range and thus the slope of the sigmoidal fit increases significantly in samples containing high Al levels (data not shown). Instead, the inflection point of the sigmoidal curve was used as it is independent of the intensity range.

As shown in FIG. 2B, the inflection point of the sigmoidal curve fit decays exponentially with increasing Al concentration, indicating that complexation and grain growth events are significant at trace levels of Al (40-60 ppb). further increase in Al (above 80 ppb) did not result in further shift of the inflection point. These results suggest that larger particles are formed in the presence of higher Al concentrations, but particle growth kinetics are unaffected above concentrations of 80 ppb. For very low concentrations (20 ppb), the inflection point was calculated to be approximately 65 hours and was therefore not captured in this experiment.

In a second study, different divalent (Ca, Mg, Zn, Ni) and trivalent (Al, Fe) cations were screened for their tendency to interact with lauric acid to form FFA salt particles. Equimolar amounts of metal ions ranging from 0 to 30 μM were used for better comparison. The conversion to ppb concentration of each is shown in Table 12.

(Table 12) Conversion of metal ion concentration

Changes in particle size (r _H ) and intensity as a function of metal cation type and concentration were evaluated over a period of 70 hours. As shown in Figure 3, particle formation and growth were observed only for trivalent metal ions (Fe, Al), whereas the presence of divalent cations (Ca, Mg, Ni, Zn) was associated with the formation of FFA salt particles. or did not result in growth. Interestingly, Al and Fe behaved quite differently. An increase in Al concentration (0-10 μM) resulted in the formation of larger grains and faster grain growth, whereas the addition of an equal amount of Fe resulted in instantaneous grain formation (r _H of 50-60 nm). , did not lead to a further increase in particle size over time. However, the scattering intensity increases with increasing Al and Fe levels, suggesting that the FFA-Fe particles do not grow over time, but the number of particles is still increasing.

For both trivalent cations, the highest concentration (30 μM) resulted in the formation of very large particles. No further size increase could be observed as the particle size was close to the upper limit of detection for DLS. Moreover, these large particles appeared to settle over time as indicated by a gradual decrease in scattering intensity (Fig. 3B).

Based on these results, the presence of divalent cations has a low risk of FFA salt particle formation in the concentration range investigated, while trivalent cations such as Al ³⁺ and Fe ³⁺ even at very low concentrations It can be concluded that it can interact with negatively charged FFAs.

Protective Effects of Chelating Agents on FFA Metal Nucleation To assess the protective effects of chelating agents, DTPA, EDTA, GLDA and PDTA were added in the range of 0-40 μM, corresponding to molar ratios of 0-10 (chelating agent to Al). was spiked into a solution containing lauric acid and 4 uM Al at different concentrations of .

As shown in Figures 4 and 5, increasing chelator concentration resulted in an overall decrease in scattering intensity and particle size, and slower particle growth over time. At a molar ratio of at least 1:1 (chelator to Al), grain formation and growth were effectively suppressed for all chelators. Small differences in efficiency between chelating agents can be attributed to their chemical structures. EDTA, GLDA and PDTA are tetraacetic acids that can complex polyvalent ions as hexadentate chelating agents, whereas DTPA has eight coordination bond-forming sites (5 carboxylate oxygen atoms and 3 pentetic acid with one nitrogen atom). The carboxylate donor group becomes increasingly protonated with decreasing pH (Eivazihollagh et al., 2017), leading to less charged chelator species and thus weaker metal complexation. Since DTPA has more donor atoms than EDTA, GLDA and PDTA, it can efficiently complex polyvalent cations at slightly acidic pH.

The protective effect of DTPA was also evaluated in the presence of lauric acid and 4 μM Fe (Fig. 6). In contrast to Al, FFA metal nucleation with Fe leads to instantaneous nanoparticle formation in the DLS assay, but no significant particle growth over time. Addition of an equimolar amount of DTPA resulted in complete suppression of grain formation and growth, similar to that of Al. At a DTPA to Fe ratio of 0.5, the scattering intensity decreased significantly, but the particle radii changed only slightly, suggesting mainly a decrease in the number of particles.

The addition of DTPA to samples containing lauric acid and actual glass leachables extracted from Exp51 borosilicate glass vials was also effective in mitigating particle formation at chelator to Al molar ratios of at least 1.5. (Fig. 7).

References Allmendinger, Andrea; Leboc, Vanessa; Bonati, Lucia; Woehr, Anne; K. Abstiens, Kathrin (2021): Glass Reachables as a Nucleation Factor for Free Fatty Acid Particle Formation in Biopharmaceutical Formulations. In: Journal of pharmaceutical sciences 110(2); 785-795. DOI: 10.1016/j. xphs. 2020.09.050.
Mahler, Hans-Christian; Gohlke, Linus; Nieto, Alejandra; Roehl, Holger; Huwyler, Joerg et al. (2018): Impact of Vial Washing and Depyrogenation on Surf ace Properties and Delamination Risk of Glass Vials. In: Pharmaceutical research 35(7); 146. DOI: 10.1007/s11095-018-2421-6.
Dixit, Nitin; Salamat-Miller, Nazila; Salinas, Paul A.; ; Taylor, Katherine D.; Basu, Sujit K.; (2016): Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles. In: Journal of pharmaceutical sciences 105(5); 1657-1666. DOI: 10.1016/j. xphs. 2016.02.029.
Doessegger, Lucette; Mahler, Hans-Christian; Szczesny, Piotr; Rockstroh, Helmut; Kallmeyer, Georg; isible particles in parental drugs. In: Journal of pharmaceutical sciences 101(8); 2635-2644. DOI: 10.1002/jps. 23217.
Doshi, Nidhi; Demeule, Barthelemy; Yadav, Sandeep (2015): Understanding Particle Formation: Solubility of Free Fatty Acids as Polysorbate 20 Degradation Byprod ucts in Therapeutic Monoclonal Antibody Formulations. In: Molecular pharmaceuticals 12(11); 3792-3804. DOI: 10.1021/acs. molpharmaceut. 5b00310.
Fish, Raphael; Padilla, Karina; Yadav, Sandeep (2020a): Evaluation of Super Refined™ Polysorbate 20 With Respect to Polysorbate Degradation, Particle Formation and Protein Stability. In: Journal of pharmaceutical sciences 109(10); 2986-2995. DOI: 10.1016/j. xphs. 2020.06.030.
Luis, Lin; Wu, Arthur; Ritchie, Kyle; Liu, Wenqiang et al. (2021): A Comprehensive Assessment of All-Oleate Polysorbate 80: Free Fatty A cid Particle Formation, Interfacial Protection and Oxidative Degradation. In: Pharmaceutical research 38(3); 531-548. DOI: 10.1007/s11095-021-03021-z.
Doshi, Nidhi; Martin, Joelle; Tomlinson, Anthony (2020b): Improving Prediction of Free Fatty Acid Particle Formation in Biopharmaceutical Drug Products: Incorporation ing Ester Distribution during Polysorbate 20 Degradation. In: Molecular pharmaceuticals 17(11); 4354-4363. DOI: 10.1021/acs. molpharmaceut. 0c00794.
Doyle Drbohlav, Laura M.; Sharma, Anant Navanithan; Gopalrathnam, Ganapathy; Huang, Lihua; Bradley, Scott (2019): A Mechanistic Understanding of Polysorbate 80 Oxidation in Histidine and Cit Rate Buffer Systems—Part 2. In: PDA journal of pharmaceutical science and technology 73(4); 320-330. DOI: 10.5731/pdajpst. 2018.009639.
Eivazihollagh, Alireza; Backstrom, Joakim; Norgren, Magnus; Edlund, Hakan (2017): Influences of the operational variables on electrochemical treatments of chelated Cu. (II) in alkaline solutions using a membrane cell. In: Journal of Chemical Technology & Biotechnology) 92(6); 1436-1445. DOI: 10.1002/jctb. 5141.
European Directorate for the Quality of Medicines: Histidine Monography 01/2017:0911. In: European Pharmacopeia 10.3 2021.
European Directorate for the Quality of Medicines: Particulate contamination: Visible particles [2.9.20]. In: European Pharmacopeia 10.0 2020.
Glucklich, Nils; Dwivedi, Mridula; Carle, Stefan; Buske, Julia; Mader, Karsten; Otherapeutic protein formulations containing sorbate 20 and sorbate 80. In: International journal of pharmaceuticals 591; 119934. DOI: 10.1016/j. ijpharm. 2020.119934.
Gopalrathnam, Ganapathy; Sharma, Anant Navanithan; Dodd, Steven Witt; Huang, Lihua (2018): Impact of Stainless Steel Exposure on the Oxidation of Polysorbate 8 0 in Histidine Placebo and Active Monoclonal Antibody Formulation. In: PDA journal of pharmaceutical science and technology 72(2); 163-175. DOI: 10.5731/pdajpst. 2017.008284.
Graf, Tobias; Abstiens, Kathrin; Wedekind, Frank; Elger, Carsten; Haindl, Markus; Proach to assess the impact of polysorbate 20 degradation on biopharmaceutical products quality in shortened time. In: European journal of pharmaceuticals and biopharmaceutics 152; 318-326. DOI: 10.1016/j. ejpb. 2020.05.017.
Hall, Troii; Sandefur, Stephanie L.; Frye, Christopher C.; ; Tuley, Tammy L.; Huang, Lihua (2016): Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A2 Isomer X1 in Monoclonal Antibody Formulations. In: Journal of pharmaceutical sciences 105(5); 1633-1642. DOI: 10.1016/j. xphs. 2016.02.022.
Hewitt, Daniel; Zhang, Taylor; Kao, Yung-Hsiang (2008): Quantitation of polyvinyl sorbate 20 in protein solutions using mixed-mode chromatography and evaporative lig ht scattering detection. In: Journal of Chromatography. A 1215(1-2), S. 156-160. DOI: 10.1016/j. chroma. 2008.11.017.
Honemann, Maximilian N.; Wendler, Jan; Graf, Tobias; Bathke, Anja; Bell, Christian H.; (2019): Monitoring polycarbonate hydrolysis in biopharmaceuticals using a QC-ready free fat acid quantification method. In: Journal of Chromatography. B, Analytical technologies in the biomedical and life sciences 1116,S. 1-8. DOI: 10.1016/j. jchromb. 2019.03.030.
Kishore, Ravuri S.; K. Fischer, Stefan; Pappenberger, Astrid; Grauschopf, Ulla; Mahler, Hans-Christian (2011a): The degradation of polymers 20 and 80 and its potential Impact on the stability of biotherapeutics. In: Pharmaceutical research 28(5); 1194-1210. DOI: 10.1007/s11095-011-0385-x.
Kishore, Ravuri S.; K. Dauphin, Isabelle Bauer; Ross, Alfred; Burgi, Beatrice; Staempfli, Andreas; Mahler, Hanns-Christian (2011b): Degradation of polysorbates 20 and 80: Studies on thermal autooxidation and hydrology. In: Journal of pharmaceutical sciences 100(2); 721-731. DOI: 10.1002/jps. 22290.
Kranz, Wendelin; Wuchner, Klaus; Corradini, Eleonora; Berger, Michelle; Hawe, Andrea (2019): Factors Influencing Polysorbate's Sensitivity Against Enzymatic Hydrolysis an d Oxidative Degradation. In: Journal of pharmaceutical sciences 108(6); 2022-2032. DOI: 10.1016/j. xphs. 2019.01.006.
Labrenz, Steven R.; (2014): Ester hydrolysis of polysorbate 80 in mAb drug product: evidence in support of the hypothesized risk after the observation of visible parts in mAb formulations. In: Journal of pharmaceutical sciences 103(8); 2268-2277. DOI: 10.1002/jps. 24054.
Lippold, Steffen; Koshari, Stijn H.; S. Kopf, Robert; Schuller, Rudolf; Buckel, Thomas; Zarraga, Isidro E.; Koehn, Henning (2017): Impact of mono-and poly-ester fractions on polycarbonate quantitation using mixed-mode HPLC-CAD/ELSD and the fluorescence micelle assembly. In: Journal of pharmaceutical and biomedical analysis 132; 24-34. DOI: 10.1016/j. jpba. 2016.09.033.
McShan, Andrew C.; Kei, Pervina; Ji, Junyan A.; Kim, Daniel C.; Wang, Y.; John (2016): Hydrolysis of Polysorbate 20 and 80 by a Range of Carboxylester Hydrolases. In: PDA journal of pharmaceutical science and technology 70(4); 332-345. DOI: 10.5731/pdajpst. 2015.005942.
Panchal, Jainik; Kotarek, Joseph; Marszal, Ewa; Topp, Elizabeth M.; (2014): Analyzing subvisible particles in protein drug products: a comparison of dynamic light scattering (DLS) and resonant mass measurement (RMM). In: The AAPS journal 16(3); 440-451. DOI: 10.1208/s12248-014-9579-6.
Hodge, Tamara; Menard, Daniele; Jerome, Richard; Ryczek, Jeff; Moore, David et al. (2019): Editate Disodium as a Polysorbate Degradation and Mono clonal Antibody Oxidation Stabilizer. In: Journal of pharmaceutical sciences 108(4); 1631-1635. DOI: 10.1016/j. xphs. 2018.11.031.
Zhou, Shuxia; Schoneich, Christian; Singh, Satish K.; (2011): Biologies formulation factors affecting metal reachables from stainless steel. In: AAPS PharmSciTech 12(1), S.E. 411-421. DOI: 10.1208/s12249-011-9592-3.

Claims (13)

A stable aqueous composition comprising a protein together with pharmaceutically acceptable excipients such as buffers, stabilizing agents including antioxidants, and further comprising at least one chelating agent. The chelating agent is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylene glycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N-carboxymethyl -N'-(2-hydroxyethyl)-N,N'-ethylenediglycine (HEDTA), ethylenediamine-N,N'-bis(2-dihydroxyphenylacetic acid) (EDDHA), 1,3-diaminopropane-N ,N,N',N'-tetraacetic acid (PDTA), N,N-bis(carboxymethyl)-L-glutamate tetrasodium (GLDA), citrate, malonate, tartrate, ascorbate, salicylic acid, aspartic acid, glutamic acid 2. The composition of claim 1, selected from the group of 3. The composition of claim 2, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). A composition according to any preceding claim, wherein the chelating agent is present in a concentration of 0.0005-2.0% (w/v). A composition according to any one of claims 1 to 4, wherein said protein is an antibody or monoclonal antibody. Use of chelating agents for the manufacture of medicaments, in particular for the manufacture of stable parenteral protein preparations or stable parenteral antibody preparations. Use of a chelating agent for the manufacture of parenteral protein or antibody preparations, wherein said parenteral protein or antibody preparations are free of visible particles over their entire approved shelf life. use of a chelating agent, characterized in that Use of chelating agents to prevent visible particle formation in parenteral protein or antibody preparations. The chelating agent is ethylene glycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N-carboxymethyl-N'-(2-hydroxyethyl)-N,N '-ethylenediglycine (HEDTA), ethylenediamine-N,N'-bis(2-dihydroxyphenylacetic acid) (EDDHA), 1,3-diaminopropane-N,N,N'N'-tetraacetic acid (PDTA), Tetrasodium N,N-bis(carboxymethyl)-L-glutamate (GLDA), citrate, malonate, tartrate, ascorbate, salicylic acid, aspartic acid, glutamic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA or pentetyl acid) and is present in a concentration ranging from 0.0005 to 2.0%. Use of a chelating agent according to claim 9, wherein said chelating agent is EDTA or DTPA. 11. Any of claims 7-10, wherein said visible particles comprise at least one polyvalent cation and free fatty acids cleaved from surfactants, such as PS20 and PS80, present in said parenteral protein preparation. Use of a chelating agent according to item 1. A pharmaceutical dosage form comprising in a container a preparation according to any one of claims 1 to 5 or a preparation obtained using a chelating agent according to any one of claims 6 to 11. . Novel compositions, methods and uses substantially as described herein.

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